Apart from sugars' proven association with dental caries and obesity, the link to cardiovascular disease, type 2 diabetes and cancer remains largely inconclusive. Most population studies rely on self-reported measures of diet, known to be associated with large measurement error, which may be obscuring the true relationship between sugars and disease risk and may explain the lack of consistency in the observed evidence. Development of novel approaches for obtaining more accurate estimates of intake is crucial for attaining more reliable risk estimates for sugars and disease risk. Recently, 24-h urinary sucrose and fructose (24uSF) was developed as a biomarker of sugars intake in two UK-based feeding studies. However, evidence on the performance and the validity of this biomarker in U.S. participants and in the context of a U.S. diet is lacking. Further, the utility of uSF measure in spot urines has never been investigated and could have significant implications for epidemiologic studies. Another promising sugars biomarker, the carbon stable isotope ratio (d13C) in red blood cell (RBCs) has been recently developed among Alaska Natives. This biomarker has been tested against self-reports only, and has never been investigated in a feeding study with known intake. We now propose a 15-d highly-controlled feeding study to comprehensively investigate the performance of 24uSF and d13C as biomarkers of sugars intake, individually and in combination, and to inform future application of these promising biomarkers among U.S. participants. The study will involve 107 men and women aged 18-70 y, consuming only foods and beverages provided by the study metabolic kitchen for 15-d, that will mimic their usual diet, assessed previously by two 7-d food records. Over the 15-d feeding period, participants will collect 8 nonconsecutive 24-h urines, and blood samples will be collected at baseline, at study completion, and 5 weeks post-study completion. First we will investigate the performance of 24uSF as a biomarker of sugars intake and we will develop a calibration equation that will define the future application of 24uSF as an unbiased measure of sugars in U.S. participants. Further, we will investigate the utility of measuring uSF in spot urin as a biomarker of intake; on two 24-h urine collection days, participants will collect urine voids n separate containers. Second, we will investigate the performance of RBCs d13C as a biomarker of sugars intake among U.S. participants under controlled conditions of a feeding study. We will also explore the use of 24-h urinary d13C as a measure of sugars intake. Third, we will investigate approaches of combining 24uSF and d13C in a unique measure. Combining these two unrelated biomarkers with different sources of error and with different time-relation to intake may provide a superior measure of intake. This study will develop validated sugars biomarkers that can be used as unbiased measures of sugars intake among U.S. individuals that will ultimately allow obtaining reliable risk estimates of sugars-disease associations. Until strong and consistent evidence for adverse health effects of sugars is found, no firm guidance can be given to the general public.